[Histonet] Nuclear artefact II

Thu Jun 28 19:43:14 CDT 2007

It has also been suggested that this nuclear bubbling could also be due
to "boiling-off" of water under the section when it is placed on a hot
plate or oven for drying (usually too soon after picking-up of the
section from the waterbath). Try allowing the sections to dry at room
temp prior to 60oC drying.

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Lee &
Peggy Wenk
Sent: Friday, 29 June 2007 10:20 AM
To: 'Gale, Nadia'; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] Nuclear artefact II

>From what I've read about nuclear bubbling, it all starts with 
>inadequate
fixation. Anything else we do to the tissue just makes it worse. Nothing
we can do will ever improve poorly fixed tissue.

If there are not enough cross-links of formalin (or any other fixative)
due to underfixation, then these few cross-links can be easily broken
later on. This causes the proteins and DNA being easily moved around
into new locations.

So some of the things that can move the proteins and DNA are:
- processing (going through alcohols and xylene will warp these under
cross-linked proteins/DNA even more)
- too much heat in processing (alcohols, xylene, paraffin)
- too long of time in hot paraffin
- too much heat during slide drying

The reason we see this artifact mostly with formalin fixed tissue is two
fold:
1) Formalin needs 1-2 days to completely fix 3 mm thick tissue sections.
In other words, we need that amount of time to create enough cross-links
so that nuclear bubbling and other protein deformities do not occur
later with processing, heat, etc.
2) Formalin is a lousy nuclear fixative. Mercury and Zinc are much
better fixatives for nuclei. And tend to fix faster than formalin. So
B5, Zenker, Zinc-Formalin are less likely to show nuclear bubbling.
However, the worst case of nuclear bubbling I ever saw (orphan annie
eyes) was in bone marrow biopsies that were underfixed in zinc formalin
(tech didn't want to wait the usual amount of time, so only let them fix
about half the time required).

So, increasing formalin fixative by one hour is NOT going to correct
this problem. It's going to take several hours increase in time in
formalin before some decrease in nuclear bubbling is seen. So leave the
tissue on the processor in the formalin as long as possible at the
beginning of the cycle, NOT in the hot paraffin at the end of the cycle.

After that, watch the temperatures during processing (keeping at 37
degrees C would be better than 42 degrees, for example for the
alcohols/xylene; keep paraffin as low as possible (2 degrees above
melting point)). Keep the temperature on the slide dryer down too. 60
degrees C for 30 minutes will be better than 80 degrees C for 15
minutes.

And tell the people grossing that they MUST cut thin sections (thickness
of a nickle = 2 mm), and NOT overstuff the cassettes. These will not
allow formalin to flow around the tissue. So the tissue won't get fixed!
So no cross-links, so no protein/DNA stabilization. Ergo, another reason
for nuclear bubbling!

Peggy A. Wenk, HTL(ASCP)SLS
William Beaumont Hospital
Royal Oak, MI 48073

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Gale,
Nadia
Sent: Thursday, June 28, 2007 12:05 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Nuclear artefact II

I've looked at everyone's suggestions/comments and am sad to report that
I cannot get rid of the nuclear "bubble" artefact from our slides.

I have tried

- decreasing water bath temp
- increasing/decreasing drying oven time before H&E staining
- varying fixation times in formalin

I am currently trying

- decreasing drying oven temperature
- changing the "wrap" that I use for the biopsies when grossing
- looking at our wax times on the processor

I agree with Bob that it's extremely frustrating that our community
cannot seem to agree on a common cause for this (what I am now
realizing) common artefact.

I'll keep you posted with my results.

Thank you so much for all of your ideas and help with this, I am most
grateful.

Sincerely,
Nadia Gale
Histotechnologist
Centre for Translational and Applied Genomics (CTAG) 3427-600 West 10th
Avenue Vancouver, BC V5Z 4E6 tel 604-877-6000 ext 2426 fax 604-877-6089
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